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Abstract

Background

Hypercholesterolemia causes inflammation and insulin resistance in the vasculature.
Previous data suggest that vascular endothelium is a physiological target of insulin.
Dyslipidemia and atherosclerosis are disorders with endothelial dysfunction that are
associated with an increased production of superoxide anion, and early deficit of
nitric oxide (NO) production. We examined alteration of plasma levels of insulin,
C-reactive protein (CRP) and total NO metabolites (NOx), as well as fatty streak formation
in the rabbit model of hypercholesterolemia.

Methods

White male rabbits were fed either a high-cholesterol diet (HC; 1% cholesterol, n = 6)
or control diet (c, n = 6) for one month. The serum levels of Cholesterol, LDL, HDL,
NOx, insulin and CRP were measured before and after study. By the end of study, rabbits'
aorta was explored for fatty streak formation.

Results

The cholesterol-rich diet induced a significant increase in total cholesterol, LDL,
and HDL as well as fatty streak lesions in HC group while there were no significant
changes of these parameters in control group (p <0.05). There was significant difference
in plasma levels of CRP, insulin and total NO metabolite between two groups of experiment.
Negative significant correlation of CRP and insulin also was observed in HC rabbits
(r = −0.99, p <0.05).

Conclusion

Parallel NOx and insulin increment and negative correlation of CRP and insulin in
HC rabbits may be suggestive a protective role of hyperinsulinemia in early atherosclerosis.

Keywords:

Introduction

Considerable data suggest vascular endothelium as a physiological target of insulin
[1,2]. In addition to the eminent metabolic effects, insulin has important vascular actions.
Insulin stimulate production of nitric oxide (NO) from endothelium by phosphatidyl
inositol 3-kinase (PI3K)-dependent signaling pathways [3]. Pathophysiologic conditions such as hyperlipidemia, and inflammation selectively
impair PI3K dependent insulin signaling pathways, creating reciprocal relationships
between insulin resistance and endothelial dysfunction (ED)[3], and ED is associated with deficit of NO production [4].

It is reported that NO has direct effects on the progression of atherosclerosis [5], but chronic inhibition of NO in the presence of a high-cholesterol diet has been
shown to induce development of atherosclerosis [6]. Furthermore, reducing endothelial NO levels is a known mechanism for inducing insulin
resistance [7]. This fact is tempting to hypothesize a potential link between insulin resistance
and atherosclerosis. It has been suggested that reduced bioavailability of NO by insulin
resistance may be an additional pathogenic factor in atherosclerosis [7,8]. The exact molecular relationship between insulin, ED and atherosclerosis is presently
unknown [9]. In order to elucidate this relationship, studying the early variation of insulin
during induction of hypercholesterolemia may help cognizance the pathogenesis of atherosclerosis.

Therefore in this study we examined plasma levels of insulin, C-reactive protein (CRP)
and total NO metabolites (NOx), as well as fatty streak formation in the rabbit model
of hypercholesterolemia.

Methods

Animals and experimental design

The study was reviewed and approved by the Ethics Committee of Isfahan University
of Medical Sciences. Twelve white male rabbits weighing between 1.9 ± 0.1 kg were obtained from the Pasteur
Institute of Iran. After 1-week acclimatization period and overnight fasting, blood
samples were taken as pre-experimental sampling. Blood samples were collected in heparinated
tubes, centrifuged (10,000 _ g), and the resulting plasma was stored at −70°C until measurement. The animals were
randomized into two groups of experiments. First group (HC group) fed rabbit chow
supplemented with 1% cholesterol, and the other group (control group) received rabbit
chow only. All animals received food and water ad libitum. After one month of experiment,
the blood samples were taken and stored again. The animals were euthanized by an overdose
of sodium pentobarbital and ex-sanguinated. The animal's aortas were harvested for
pathological investigation. Triglyceride, total cholesterol, high density lipoprotein
(HDL) cholesterol and, low density lipoprotein (LDL) levels were measured using a
standard enzymatic kit (Pars Azmoon Co, Iran). NOx measurement was done by total NO/nitrite/nitrate assay kit (R&D Systems, Minneapolis,
USA) which based on the enzymatic conversion of nitrate to nitrite by nitrate reductase
as previously described [10]. CRP (IBL Co, Germany) was measured using an enzyme-linked immunosorbent assay kit
according to manufacturer’s instruction. Insulin was measured by the electrochemiluminescence
immunoassay method using the Elecsys insulin reagents kit(Roche Diagnostics, Mannheim,
Germany) on an automated Roche Modular Analytics Module E170 (Roche Diagnostics).
The detection limit was 0.2 pmol/L.

Pathologic investigation

The entire aortas, from the aortic arch to the external iliac arteries, were fixed
in buffered 10% formalin for 24 h, and then embedded in paraffin. The paraffin-embedded
specimens were sectioned at 5 μm. 20 sections were stained with haematoxylin and eosin,
and examined by light microscopy for the fatty streak lesions formation and the intima
and media thickness of aorta measurement. Then the intima / media thickness (IMT)
ratios were calculated as the atheroma formation index for each animal.

Statistical analysis

The data are reported as the mean ± SEM. A statistical software package, SPSS (version
13), was used to perform statistical analysis. The data were tested for normality
and homogeneity of variance. Paired and unpaired Student's t-test was used to assess the significance of any changes within and between groups
respectively. Statistical significance was accepted at p <0.05.

Results

The cholesterol-rich diet induced a significant increase in total cholesterol, LDL-cholesterol,
and HDL-cholesterol in hypercholesterolemic group while there were no significant
changes of these parameters in control group. There was significant difference in
total cholesterol, LDL-, and HDL between two groups of experiment (Table 1).

Table 1.The serum levels of total cholesterol, LDL, HDL, Insulin in two groups of the study
at baseline and after experiment

There was no significant difference in plasma level of insulin before and after experiment
in control group (p <0.05) while the insulin level was significantly increased in
case group (Table 1). There was significant difference in plasma levels of insulin between two groups
of experiment (p <0.05) (Table 1).

Similarly, there was no significant difference in plasma level of total NO metabolite
before and after experiment in control group while the total NO metabolite level was
significantly increased (p <0.05) (Figure 1). There were significantly more plasma levels of total NO metabolite in cases compare
to control group (p <0.05) (Figure 1).

Figure 1 .Total nitric oxide metabolite (μmol/l) levels before and after of experiment in case
and control groups. Paired and unpaired Student’s t-test was used to assess the significance of parameter
changes within and between groups respectively. (P <0.05).

The plasma level of CRP significantly increased in case group (p <0.05) (Figure 2) whereas there was No significant difference in plasma level of CRP, insulin and
total NO metabolite were found before and after experiment in control group (Figure
2). There was significant increased plasma levels of CRP in cases compare to control
group (p <0.05) (Figure 2).

Figure 2 .C-reactive protein levels before and after of experiment in case and control groups. Paired and unpaired Student’s t-test was used to assess the significance of any changes
within and between groups respectively (P <0.05).

At the end of study, there was no fatty streak lesions in the control group aortas
while the IMT ratio was 0.27 ± 0.1 in HC group (p = 0.05).

There was a significant negative correlation between plasma levels of insulin and
CRP concentration (r = −0.99, p = 0.03) by the end of experiment in control group.

Discussion

This study was designed to investigate the insulin alteration as a consequence of
hypercholesterolemic diet in rabbit model of early atherosclerosis. The cholesterol-rich
diet resulted in a significant increase in total cholesterol, LDL-cholesterol, and
HDL-cholesterol as well as the development of early lesions that were representative
of fatty streak initiation similar to those in humans. Thus, the model allowed us
to examine some factor alteration during the initiation of atherosclerosis. In current
experiment, CRP significantly increased in hypercholesterolemic rabbits while there
was no significant change in control group.CRP has not only been proposed as risk
factors of cardiovascular disease, but has also been associated with the variables
of insulin resistance syndrome [11]. Since hypercholesterolemia is a major participator in the inflammatory process of
atherosclerosis, several in vitro and in vivo studies have been showed the increased level of CRP in hypercholesterolemic animals
and patients [12,13].

As the results of our study indicated, 30 days of cholesterol-feeding enhanced the
content of total nitrite, as has been demonstrated previously [14-16]. It has been proposed that enhanced NO synthesis might be a defense mechanism to
compensate for continuous inactivation of NO by oxygen-derived free radicals [17,18].Another proposed mechanisms responsible for the elevation of nitrite may be NO production
by other isoforms of Nitric Oxide Synthase (NOS)enzymes. Increased NOS mRNA and protein
of atherosclerotic vessels reported in other experiments showing that aortas of hypercholesterolemic
rabbits release larger quantities of nitrogen oxides than do normal vessels in early
atherosclerosis [19].

Interestingly, in parallel to total NO metabolite alteration, the hypercholesterolemia
induced insulin increment in rabbits. At the cellular level, it has been shown that
exposure to insulin increases eNOS mRNA and protein synthesis [20,21]. Furthermore, insulin increased eNOS production through increasing the activity of
AP-1, a transcription factor that bind to the eNOS promoter [20].

Furthermore, in humans, infusion of insulin causes NO dependent vasodilatation and
increased blood flow [22]. In both humans [23] and in animal models of insulin resistance [24], there is a specific impairment of PI3K-dependent signaling pathways. Thus, insulin
resistance would be associated with a decrease in eNOS phosphorylation and decreased
endothelial NO production. So, it seems that early hyperinsulinemia in the beginning
of the atherosclerosis may be a protective mechanism for endothelial function. However,
it has been shown that prolonged exposure of endothelial cells to high insulin levels
induces a downregulation of the PI3K/Akt/eNOS axis. Such impairment of insulin signaling
in prolonged hyperinsulinemia may result in ED and promote atherogenesis [25].

In this study, there was a significant negative correlation between plasma levels
of insulin and CRP concentration several experiments demonstrated the anti-inflammatory
effects of insulin [26-30]. It has been showed that insulin suppressed the expression of the pro-inflammatory
intracellular adhesion molecule (ICAM)-1, the chemokine, monocyte chemoattractant
protein-1 (MCP-1), and the key pro-inflammatory transcription factor, nuclear factor
[kappa]B (NF[kappa]B) in human aortic endothelial cells at physiologically relevant
concentrations [26,27]. In patients with acute myocardial infarction, insulin also suppressed C-reactive
protein (CRP) and serum amyloid A (SAA) by 40% within 24 h of the start of the insulin
infusion while glucose concentrations rendered unchanged [28]. This effect of insulin was corroborated in patients with myocardial infarction as
well as in patients undergoing coronary artery bypass grafts in two studies [28-30].

In summary, insulin may promotes endothelial function, through increased NO production,
which may has anti-inflammatory effects and cause slower atherosclerotic progression.

Competing interests

The authors have no competing interests.

Authors’ contributions

SHJ had substantial contributions to conception and design of the study, analysis
of the data and drafting the manuscript. MN had substantial contributions to conception
of the study, analysis of the data. AF did the acquisition of data and analysis. ND
participated in its coordination and helped to draft the manuscript. All authors have
read and approved the content of the manuscript.

Acknowledgement

This study was supported by Isfahan University of Medical sciences, Isfahan, Iran
(Grant #386327) and physiology research center.